Conflict of interest statement

Figures

Identification of siRNA targets. (A) Schematic representation of lentiviral screen for tumorigenic signature using the Open Biosystems GIPZ vector system. shRNAs targeting 477 genes along with controls were plated onto 14 × 96-well plates for a total of 1,128 shRNA in SUM159 and BT549 cell lines. (B) Increasing concentration of positive control Notch inhibitor (GSI) added to SUM159 cells to demonstrate a dose-dependent decrease in mammosphere forming efficiency. (C) Identification of top targets using Z score analysis of data in SUM19 and BT549 cell lines. Activity in both cell lines is color-coded, with bold red circles denoting reduced MSFE in both lines, and open red and gray circles denoting reduced MSFE in SUM159 and BT549, respectively; others are nonsignificant. (D) List of five genes identified by Z score analysis. (E) Validation of BCSC targets was conducted with a low titer of virus (pGIPZ vector) at a multiplicity of infection of 10 using MSFE. (F) Secondary MSFE with siRNA (50 nM) from the sequence derived from pGIPZ vector shRNA against MLF2 and RPL39 in three triple negative cell lines, MDAMB231, SUM159, and BT549. Data analyzed by one way ANOVA and plotted as mean + SEM for n = 6 replicates; *P < 0.05.

Fig. 2.

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In vivo treatment of primary…

Fig. 2.

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In vivo treatment of primary cancer and lung metastasis xenografts with RPL39 and…

Increase in wound healing, MSFE, and proliferation with RPL39 and MLF2 overexpression. (…

Fig. 3.

Increase in wound healing, MSFE, and proliferation with RPL39 and MLF2 overexpression. (A) Representative image of MDAMB231 and BT549 cells treated with vehicle, RPL39, and MLF2. Overexpression of RPL39 and MLF2 genes demonstrates a statistically significant increase in the migration index of these cells. (B) The % secondary MSFE was determined with overexpression of RPL39 and MLF2 plasmid DNA in three triple negative breast cell lines, SUM159, BT549, and MDAMB231, in mammosphere growth media. Data were analyzed at 72 h. (C) A dose-dependent increase in proliferation was observed upon 1 µg and 5 µg of plasmid DNA transfection in the three breast cancer cell lines.

RPL39 and MLF2 affect nitric oxide synthase (NOS) signaling. (A) Mutual exclusivity analysis of RPL39 and MLF2 in TCGA breast-cancer database demonstrates a statistically significant chance of co-occurrence odds ratio > 10 (P = 0.02). These data show strong tendency of co-occurrence for these two genes in breast cancer. (B and C) Microarray analysis of RPL39 and MLF2 siRNA, respectively, compared with scrambled siRNA in patient-derived xenografts BCM2665, at P < 0.005 and fold change >1.5. (D) Western analysis of control vs. overexpression of RPL39/ MLF2 shows an increase in both iNOS and eNOS with no changes in nNOS in two triple negative cell lines. (E) Western analysis of siRNA-treated samples comparing scrambled siRNA vs. MLF2/RPL39 siRNA, in vitro in three cell lines, SUM159, BT549, and MDAMB231, shows decrease in eNOS and iNOS, with no substantial change in nNOS. (F) Wound-healing assays were performed to confirm the role of NOS signaling in RPL39 and MLF2 overexpressing cell lines, using NOS inhibitor l-NAME. This inhibition was rescued by addition of three molar excess l-arginine.

Fig. 5.

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RPL39 and MLF2 expression is…

Fig. 5.

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RPL39 and MLF2 expression is altered by hypoxia. Two breast cancer cell lines…

Fig. 5.

RPL39 and MLF2 expression is altered by hypoxia. Two breast cancer cell lines (SUM159 and MDAMB231) were grown under hypoxic conditions (1% O2) vs. normoxic conditions for 24 h. (A) These cell lines were analyzed for the two genes (MLF2 and RPL39), NOS signaling pathway (iNOS and eNOS) (Right). HIF1α levels were assessed by ELISA (Left). (B) Inhibition of iNOS under normoxia by 1400W reduced HIF1α, RPL39, and MLF2. Additionally, downstream effectors of NOS signaling SGC and PKG-1 were also reduced. Overexpression of RPL39 and MLF2 in these cell lines elevated SGC and PKG-1 levels. (C) RPL39 and MLF2 genes were overexpressed in SUM159 and MDAMB231 cells and exposed to hypoxia (1% O2) for 1 d followed by analysis of HIF1α levels by ELISA. (D) GSEA analysis of HIF1α-dependent hypoxia in patient-derived BCM2665 xenografts treated with siRNA against RPL39 and MLF2. (E) Tabular representation of HIF1α-dependent hypoxia-related genes that are significantly altered by siRNA against RPL39 and MLF2 from the same samples. (F) Diagram of cross-talk between iNOS, HIF1α, and RPL39/MLF2.

Fig. 6.

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Identification and validation of damaging…

Fig. 6.

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Identification and validation of damaging mutations in lung metastasis and sorted BCSC population.…